1. Field of the Invention
The present invention relates to the development of an electrochemiluminescence (ECL) based enzyme immunoassay for the detection and the quantitative measurement of analytes. The immunoassay is based on a catalytic process employing xcex2-lactamase-conjugated anti-analytes which enzymatically hydrolyze electrochemiluminescent substituted substrates, making them strongly electrochemiluminescent. The immunoassay is very sensitive and is suitable for the detection and monitoring of any analyte for which an anti-analyte can be made.
2. Description of Related Art
An ever-expanding field of applications exists for rapid, highly specific, sensitive, and accurate methods of detecting and quantifying chemical, biochemical, and biological substances, including enzymes such as may be found in biological samples. Because the amount of a particular analyte of interest such as an enzyme in a typical biological sample is often quite small, analytical biochemists are engaged in ongoing efforts to improve assay performance characteristics such as sensitivity.
One approach to improving assay sensitivity has involved amplifying the signal produced by a detectable label associated with the analyte of interest. In this regard, luminescent labels are of interest. Such labels are known which can be made to luminesce through photoluminescent, chemiluminescent, or electrochemiluminescent techniques. xe2x80x9cPhotoluminescencexe2x80x9d is the process whereby a material luminesces subsequent to the absorption by that material of light (alternatively termed electromagnetic radiation or emr). Fluorescence and phosphorescence are two different types of photoluminescence. xe2x80x9cChemiluminescentxe2x80x9d processes entail the creation of the luminescent species by a chemical reaction. xe2x80x9cElectrochemiluminescencexe2x80x9d is the process whereby a species luminesces upon the exposure of that species to electrochemical energy in an appropriate surrounding chemical environment.
The signal in each of these three luminescent techniques is capable of very effective amplification (i.e., high gain) through the use of known instruments (e.g., a photomultiplier tube or pmt) which can respond on an individual photon by photon basis. However, the manner in which the luminescent species is generated differs greatly among and between photoluminescent, chemiluminescent, and electrochemiluminescent processes. Moreover, these mechanistic differences account for the substantial advantages as a bioanalytical tool that electrochemiluminescence enjoys vis a vis photoluminescence and chemiluminescence. Some of the advantages possible with electrochemiluminescence include: (1) simpler, less expensive instrumentation; (2) stable, nonhazardous labels; and (3) increased assay performance characteristics such as lower detection limits, higher signal to noise ratios, and lower background levels.
As stated above, in the context of bioanalytical chemistry measurement techniques, electrochemiluminescence enjoys significant advantages over both photoluminescence and chemiluminescence. Moreover, certain applications of ECL have been developed and reported in the literature. U.S. Pat. Nos. 5,147,806, 5,068,808, 5,061,445, 5,296,191, 5,247,243, 5,221,605, 5,238,808 and 5,310,687, the disclosures of which are incorporated herein by reference, detail certain methods, apparatuses, chemical moieties, inventions, and associated advantages of ECL.
A particularly useful ECL system is described in a paper by Yang et al., Bio/Technology, 12, pp. 193-194 (Feb. 1994). See also a paper by Massey, Biomedical Products, October 1992 as well as U.S. Pat. Nos. 5,235,808 and 5,310,687, the contents of these papers and patents being incorporated herein by reference.
ECL processes have been demonstrated for many different molecules by several different mechanisms. In Blackburn et al. (1991) Clin. Chem. 37/9, pp. 1534-1539, the authors used the ECL reaction of ruthenium (II) tris(bipyridyl), Ru(bpy)32+ are very stable, water-soluble compounds that can be chemically modified with reactive groups on one of the bipyridyl ligands to form activated species with which proteins, haptens, and nucleic acids are readily labeled.
Beta-lactamases which hydrolyze the amide bonds of the xcex2-lactam ring of sensitive penicillins and cephalosporins are widely distributed amongst microorganisms and play a role in microbial resistance to xcex2-lactam antibiotics. Beta-lactamases constitute a group of related enzymes which are elaborated by a large number of bacterial species but not by mammalian tissues and can vary in substrate specificities. See generally Payne, D. J., J. Med. Micro (1993) 39, pp. 93-99; Coulton, S. and Francois, 1., Prog. Med. Chem. (1994) 31, 297-349; Moellering, R. C., Jr., J. Antimicrob. Chemother. (1993) 31 (Suppl. A), pp. 1-8; and Neu, H. C., Science (1992) 257, pp. 1064-1072.
Several methods currently exist for the detection of microbial xcex2-lactamases. Some representative examples follow.
W. L. Baker, xe2x80x9cCo-existence of xcex2-lactamase and penicillin acylase in bacteria; detection and quantitative determination of enzyme activitiesxe2x80x9d, J. Appl. Bacteriol. (1992) Vol. 73, No. 1, pp. 14-22 discloses a copper-reducing assay for the detection of penicilloates and fluorescamine assay to detect 6-aminopenicillanic acid concentrations when both substances were produced by the action of the enzymes on a single substrate.
U.S. Pat. No. 5,264,346 discloses a calorimetric assay for xcex2-lactamase which has a variety of applications. The assay is based on the decolorization of a chromophore formed by oxidation of either the N-alkyl derivative of p-phenylenediamine or the 3,3xe2x80x2,5,5xe2x80x2-tetraalkyl derivative of benzidine. The decolorization is attributed to the presence of an open xcex2-lactam ring product resulting from the hydrolysis of cephalosporin or penicillin. Decolorization with the open xcex2-lactam product of penicillin requires the presence of a decolorization enhancer such as mercury containing compounds. The enhancer is not required for decolorization with the open xcex2-lactam product of cephalosporin.
U.S. Pat. No. 4,470,459 discloses a rapid method for the detection of the presence of xcex2-lactamase from microbial sources which is based on a xcex2-lactamase conversion of a xcex2-lactam substrate which reverses its ability to fluoresce. Specific xcex2-lactams mentioned as having this property include ampicillin, cephalexin, amoxicillin, cefadroxil and cephaloglycin. The change in the ability to fluoresce is attributed to the presence of xcex2-lactamase.
WO 84/03303 discloses a microbiological test process for identifying producers of xcex2-lactamase. The assay relies on changes in acidity which affect the fluorescence of the indicator such as coumarin. This change in acidity is attributed to the conversion product produced by the presence of the xcex2-lactamase.
A. C. Peterson et al., xe2x80x9cEvaluation of four qualitative methods for detection of xcex2-lactamase production in Staphylococcus and Micrococcus speciesxe2x80x9d, Eur. J. Clin. Microbiol. Infect. Dis. (1989), Vol. 8, No. 11, pp. 962-7 presents certain factors which were employed in evaluating qualitative assays for xcex2-lactamase.
Robert H. Yolken et al., xe2x80x9cRapid diagnosis of infections caused by xcex2-lactamase-producing bacteria by means of an enzyme radioisotopic assayxe2x80x9d, The Journal of Pediatrics, Vol. 97, No. 5 (November 1980) pp. 715-720 discloses a sensitive enzymatic radioisotopic assay for the measurement of xcex2-lactamase as a rapid test for detection of bacterial infection. The assay protocol involves an incubation step with sample followed by the separation step on a positively charged column such as DEAE-Sephacel prior to measurement of the radioactivity of eluted fractions. The xcex2-lactamase converted penicillinic product has an additional carboxyl group which insures its stronger binding to the positively charged column than the penicillin. Differences in radioactivity between the eluted fractions and the original values are attributed to the presence of xcex2-lactamase.
In immunoassays generally, antibodies (equivalently referred to herein as xe2x80x9canti-analytesxe2x80x9d) are used to detect analyte. Commonly, an anti-analyte is labeled with a molecule that is detectable by, for example, absorbance, fluorescence, luminescence, or electrochemiluminescence. Alternatively, the antibody can be labeled with an enzyme that creates or destroys a compound with one of these features. There are two main types of enzyme immunoassays; enzyme-linked immunosorbant assays (ELISA) and enzyme-multiplied immunoassay techniques (EMIT). S. C. Anderson and S. Cockayne, Clinical Chemistry: Concepts and Applications, W. B. Saunders (1993) Philadelphia, Pa. In enzyme immunoassays, the process is catalytic such that multiple detectable labels are formed, giving the possibility of enhanced sensitivity.
Electrochemiluminescence (ECL) immunoassays are conventionally carried out with antibody conjugated to the label, which is generally a derivative of tris(bipyridyl) ruthenium(II) (abbreviated as Ru(bpy)32+) G. Blackburn et al. (1991) Clin. Chem. 37, 1534-1539. In these assays, every antibody has a limited number of Ru(bpy)32+ molecules on its surface (for example, 6-8).
Compositions and methods have now been discovered for the preparation and uses of xcex2-lactamase-conjugated antibodies in ECL-based immunoassays. For example, the enzyme xcex2-lactamase can efficiently hydrolyze Ru(bpy)32+ substituted penicillins. The penicillins, termed Ru-Amp and Ru-APA, are only very weakly electrochemiluminescent, but when they are hydrolyzed by xcex2-lactamase according to the present invention they become strongly electrochemiluminescent. The presence of xcex2-lactamase therefore can be detected with a high level of sensitivity in an ECL instrument using either of these compounds. As opposed to conventional ECL immunoassays where the Ru(bpy)32+ label is directly attached to the antibody, in the enzyme-based ECL immunoassays of the present invention, the electrochemiluminescently-active ruthenium complex is catalytically generated by the enzyme attached to the antibody surface. Thus, instead of one antibody permitting a few (typically 6-8) ruthenium labels to generate light, one antibody-enzyme complex can generate typically 2000 ruthenium labels per second and could generate as many as 10,000 or more.
Conventional ECL-based immunoassays employ ruthenium labeled antibodies. In the present invention, an immunoassay has been discovered in which the ruthenium-labeled antibody is replaced with an enzyme-labeled antibody. The enzyme is xcex2-lactamase. Tripropylamine (TPA) or similar reductants are omitted from the solution and, for example in the case of infection related assays, ruthenium-labeled penicillins are used instead. In the presence of xcex2-lactamase-labeled antibody, the ruthenium labeled substrates are catalytically hydrolyzed, generating an enormous increase in ECL. The assay is superior to the use of ruthenium-labeled antibody immunoassays because enzyme-generated ECL-active ruthenium is a catalytic process, forming many ECL active molecules.
Broadly stated, the invention contemplates an electrochemiluminescence based immunoassay for the detection of analytes. The invention employs enzymes such as xcex2-lactamases, proteases or oxido-reductases conjugated to antibodies and ECL labels and enzyme substrates, preferably ECL label substituted substrates such as ECL label substituted antibiotics, peptides, and nicotinamide adenine dinucleotide (NADH) which together provide an antibody-enzyme complex which can catalytically generate up to thousands of ECL active labels per second.
Central to use of electrochemiluminesence methodology as a measuring system for analytes was the recognition that xcex2-lactamase can efficiently hydrolyze Ru(bpy)32+ substituted penicillins. The penicillins, Ru-Amp and Ru-APA, are only very weakly electrochemiluminescent but when they are hydrolyzed by xcex2-lactamase they become strongly electrochemiluminescent.
Various assay formats can be employed in the practice of the invention as will be apparent to those skilled in the art. These include a sandwich assay using, for example, magnetic beads or other solid support such as carbon fibrils, a competitive assay using antigen conjugated to free xcex2-lactamase, a competitive assay where the xcex2-lactamase is a recombinant protein containing a segment that is bound by an antibody that also binds the chosen analyte wherein the enzyme is inactivated by antibody binding, and ELISA where xcex2-lactamase is a reporter on a secondary antibody. The Immunoassay Handbook, D. Wild, Ed. (1994) Stockton Press, New York.